PREreview of Trifluoroacetate reduces plasma lipid levels and the development of atherosclerosis in mice

This review resulted from the graduate-level course "How to Read and Evaluate Scientific Papers and Preprints" from the University of São Paulo, which aimed to provide students with the opportunity to review scientific articles, develop critical and constructive discussions on the endless frontiers of knowledge, and understand the peer review process.

Summary

TFA is commonly used as a carrier or counterions accompanying synthetic peptides. Previous works have attributed to synthetic peptides that mimic apolipoproteins like apoA-I and apoE, reduce the plasma lipid levels and atherosclerosis in mice through PPAR-α-mediated peroxisome proliferation, with effect across different mouse models and administration routes (i.p. and oral). This study suggests that TFA, rather than the peptides themselves, may have influenced previous research outcomes, raising concerns about its widespread exposure through pharmaceuticals and industrial processes. Additionally, the study underscores TFA’s potential as a therapeutic agent for atherosclerosis, highlighting the need for further research to explore its biological effects and possible clinical applications.

Overall, the article is well written, making it easy to read even for readers from other fields. In particular, the description of animal strains and the reasons for their choice is very useful for non-experts.

The study is noticeably detailed, and focuses on clear questions: is TFA responsible for the effects observed in treatments with peptides carried with TFA? How and how much? The answers to these questions are satisfactorily presented, with very few points that deserve to be cleared out.

Major issues

1) Animal gender, strains and feeding

• In the “TFA induces peroxisome proliferation in vivo and in cell culture” section, is there a reason to compare females fed with high-fat diet with males fed with CHOW diet? Why did the authors not compare HFD fed females with CHOW fed females, and HFD fed males with CHOW fed males instead? 

• Some experiments were conducted using both males and females, but only in the LDLr-/- model. Why was this approach not applied to the other experimental models? What was the criterion for doing this only in this model? Additionally, bile acid quantification in plasma and feces was conducted only in male mice. Were the same experiments performed in females? It would be interesting to present these results as well.

• In Figure 2, why is the last graph based on male mice, while the first two parts were conducted with females?

• A significant reduction in atherosclerotic lesions was observed in apoE-/- mice, but only in males. The reason for this gender difference is not clearly explained or discussed.

• The authors mention later in the “TFA treatment induces peroxisome proliferation and exerts anti-atherosclerotic effects in apoE-/- mice” and “TFA induces peroxisome proliferation in wild-type C57B1 mice” sections that there were not observed TFA-induced reductions of plasma cholesterol or triglyceride levels in apoE-/- animals and noticed lowered triglycerides and cholesterol levels in wild-type mice. According  to the group’s interpretation, it seems that the results represented in Figure 1 are the most relevant, guiding the rest of the work. Could the authors explain why these experiments were not performed with the other two mice strains and only with LDLr-/-?

• Why were different exposure times adopted across the various models? Additionally, why is the TFA dosage lower (130 µmol/kg/day) in the wild-type model?

2) Peroxisome proliferation

• The study hints at TFA's mechanism of action through PPAR-α and peroxisome proliferation activation. However, this conclusion needs further investigation to be confirmed, or at least to require a stronger base to sustain the proposed mechanism (i.e., whether it acts as a direct agonist or through an indirect pathway). As it’s known, lipids are also directed to the mitochondria for B-oxidation: Did the authors obtain any data regarding this organelle when carrying out this work? It has been shown that mitochondrial dysfunctions are associated with the initiation and progression of atherosclerosis by elevating the production of ROS (Suáres-Rivero et. al., 2021), so maybe it would be interesting to look into this organelle as well. This could help to elucidate TFA’s mechanism of action inside the body. Overall, it would be interesting to analyze β-oxidation and lipid biosynthesis pathways to assess key enzymes:

  -)FASN and ACC (lipid biosynthesis),

  -)CPT1 and CPT2 (mitochondrial function),

  -)SREBP1c (lipid synthesis regulator).

  Assessing both expression levels and enzyme quantity, would provide critical insights into the underlying mechanisms. 

• The article would benefit from a conclusion section to summarize TFA’s effect and to propose a mechanism by adding a simple scheme to help understanding. It would help the readers to understand all conclusions and perspectives that the authors reached in this work.

  Minor issues

  1) Formatting

• Although acceptable in some cases, the use of the first person (we, our, etc.) should be avoided to give a more focused and objective tone to the report itself and prevent claims of biased interpretation of data.

• In the results section, the titles of each sub-section should be separated from the first paragraph for a better readability and aesthetics of the document overall. The article would benefit from adding space between each result section for a cleaner look, resulting in a text less tiresome for the reader to follow since you have many results and long texts but with spacing for them to catch a break and process.

• Proper use of capital letters:

  -) Figure 1: capitalize “PBS” in “pbs vehicle” in the x axis.

  -)Every first letter in the titles of figures and graphs (e.g. “Time post administration (h)” instead of “time post administration”), “Female” not “female”, “Plasma cholesterol (mg/mL)” not “plasma cholesterol”) should be capitalized.

• Make sure all results presented in images show how many biological replicates were made to give the reader of this article a better understanding of the impact/representativity of the data obtained and analytical rigor of the experiments.

2) Presentation

• The article would benefit from an introductory image or scheme in the introduction section, maybe summarizing the experimental process (mice with atherosclerosis plaques treated with TFA peptides, induction of peroxisome proliferation, atherosclerosis reduction, etc). This would contribute to making the article more appealing and clearer. It would give the reader a glint of what is coming throughout the text.

• The article would benefit from a last paragraph in the introduction section summarizing which are the main findings and reinforcing the main goal of the work. It would help the readers to follow a straight narrative of what is being reported in a more fluid way.

Final comment

This work-group would like to acknowledge the authors for the opportunity of reading such a complete, detailed and, most importantly, accessible study. The group also hopes these comments will be perceived as contributions intending to improve this work even more.

Competing interests

The authors declare that they have no competing interests.